Project Summary Cortical inhibitory cells are critical for regulating information processing and synaptic plasticity in neural circuits. This plasticity is essential for learning and memory, and is an important feature of the auditory cortex, especially for learning the significance of sensory signals such as speech. Long-term synaptic plasticity requires sensory experience and activation of neuromodulatory systems such as the cholinergic nucleus basalis, which conveys behavioral context to local cortical circuits. However, little is known about how cortical interneurons are involved in these mechanisms, or if different inhibitory cell types have different roles for developmental or adult plasticity. Recently we developed an approach to measure long-term excitatory and inhibitory synaptic modifications in vivo over hours to weeks. These experiments revealed that prior to experience with sounds, cortical inhibition was initially mismatched with excitation, but becomes `balanced' with excitation after experience or training. [These experiments now allow us to construct a new framework for understanding the roles of 5HT3aR and non-5HT3aR cortical interneurons during auditory behavior in mice, with a series of behavioral, imaging, and recording experiments integrated with the larger collaborative PPG structure. We hypothesize that there are important functional differences in these cell types, in terms of their relative contributions to auditory behavior (Aim 1), cholinergic modulation (Aim 2), and cortical microcircuit organization and plasticity (Aim 3). Specifically, in Aim 1 we will first examine the behavioral relevance of specific cortical interneuron subtypes, as initially-naive mice are trained to perform an auditory detection and recognition task we have used in the lab for years. We ask how sensory experience and behavioral training might recruit these cell types and naturally shape excitatory and inhibitory circuit elements, using whole-cell recordings combined with 2-photon Ca2+ imaging to directly measure excitation and various cell-type-specific sources of inhibition in vivo. In Aim 2 we examine if these cell types are differentially affected by cholinergic modulation, perhaps due to differential sensitivity to acetylcholine or specific wiring of cholinergic input into cortex. Finally, in Aim 3 we will make recordings in cortical brain slices, to document how different cortical interneuron types are synaptically connected and modified for circuit operation.] In summary, here we will use in vivo and in vitro electrophysiology, imaging, and optogenetics to ask how different cortical interneurons (5HT3aR vs non-5HT3aR) govern sensory processing and plasticity. The two core concepts of these studies involve long-term synaptic plasticity, believed to be a major neural correlate of learning and memory, and excitatory-inhibitory balance- the precise regulation of excitation by inhibitory circuits. These processes are believed to be disrupted in a large number of neurological conditions and mental health disorders, highlighting an urgent need for a more complete description of cortical organization and function during behavior.